Ionizing radiation is useful in the treatment of cancer and for ablation of pathologic tissues because of the cytotoxic effects which result from persistent DNA double strand breaks or activation of program cell death (Haimovitz-Friedman et al., 1994; Garcia-Barros et al., 2003; Brown & Attardi, 2005). Radiation causes rapidly proliferating cells, such as tumor and cancer cells, to undergo cell death by apoptosis, both in vivo and in vitro (Antonakopoulos et al., 1994; Li et al., 1994; Mesner et al., 1997).
Current radiation therapy is frequently unsuccessful at completely eradicating cancer cells from a patient, however. This is true for at least two reasons. One reason cancer can recur is that it is often not possible to deliver a sufficiently high dose of local radiation to kill tumor cells without concurrently creating an unacceptably high risk of damage to the surrounding normal tissue. Another reason is that tumors show widely varying susceptibilities to radiation-induced cell death. Ionizing radiation activates pro-survival response through phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signal transduction pathways (Dent et al., 2003; Tan & Hallahan, 2003; Tan et al., 2006; Yacoub et al., 2006). PI3K catalyzes the addition of a phosphate group to the inositol ring of phosphoinositides normally present in the plasma membrane of cells (Wymann & Pirola, 1998). The products of these reactions, including phosphatidyl-4,5-bisphosphate and phosphatidyl-3,4,5-trisphosphate, are potent second messengers of several RTK signals (Cantley, 2002). In vitro studies have indicated that PI3K and Akt are involved in growth factor-mediated survival of various cell types (Datta et al., 1999), including neuronal cells (Yao & Cooper, 1995; Dudek et al., 1997; Weiner & Chun, 1999), fibroblasts (Kauffmann-Zeh et al., 1997; Fang et al., 2000), and certain cells of hematopoietic origin (Katoh et al., 1995; Kelley et al., 1999; Somervaille et al., 2001).
The initial molecular events that trigger radiation-induced Akt signal transduction are presently unknown. The triggering events could involve hydroxyl interaction with some membrane lipids, signaling proteins or DNA (Kolesnick and Fuks 2003; Kufe and Weichselbaum 2003; Lammering et al. 2004; Truman et al. 2005).
Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that normally acts directly on vascular endothelium to promote the survival of newly formed vessels (Alon et al., 1995; McMahon, 2000). VEGF has also been implicated in tumor proliferation (Bell et al., 1999), and several transformed cell lines express unusually high levels of VEGF (Kieser et al., 1994; Grugel et al., 1995; Graeven et al., 1999). In addition, elevated VEGF expression is clinically relevant as it is associated with worsened prognosis (Valter et al., 1999).
Elevated VEGF levels also correlate with radiation stress and radiotherapy resistance (Shintani et al., 2000). For example, VEGF expression is elevated in such radioresistant tumors as malignant glioma and melanoma (Liu et al., 1995). Interfering with VEGF signal transduction increases the in vitro radiosensitivity of glioblastoma and melanoma tumor models (Geng et al., 2001). These data suggest a role for VEGF in promoting cellular survival following radiotherapy. The mechanisms by which VEGF exerts this protective effect have not been elucidated, however.
Both in vitro and in vivo experiments have suggested that VEGF expression is induced when cells or tumors are exposed to ionizing radiation (Katoh et al., 1995; Gorski et al., 1999). For example, when growing Lewis lung carcinoma (LLC) cells are treated in vitro with different doses of irradiation, VEGF levels showed a dose-dependent increase within 24 hours of treatment (Gorski et al., 1999). Several other human tumor cell lines also showed an increase in VEGF expression after in vitro exposure to radiation, including Seg-1 (esophageal adenocarcinoma), SQ20B (a radioresistant squamous cell carcinoma line), U1 (melanoma), and T98 and U87 (glioblastoma; Gorski et al., 1999). Tumors produced in vivo by implanting LLC, Seg-1, or SQ20B cells into mice also showed enhanced VEGF expression after exposure to radiation (Gorski et al., 1999).
The induction of VEGF expression is associated with increased radioresistance of these cells and tumors. Neutralizing antibodies to VEGF, a soluble extracellular component of the Flk-1 receptor (one of three VEGF receptors so far identified), and a Flk-1-specific inhibitor are all able to eliminate this resistance phenotype both in vitro and in vivo, presumably by interfering with the interaction of VEGF with its receptor(s) (Gorski et al., 1999; Geng et al., 2001). Currently, however, effective strategies for enhancing the radiosensitivity of tumors in vivo by interfering with VEGF signal transduction are not available.
Recent evidence suggests that the cellular survival pathways involving VEGF and PI3K/Akt might overlap. For example, neovascular endothelial cells upregulate the expression of platelet-derived growth factor β receptors (βPDGFRs) during such processes as wound healing, inflammation, and glioma tumorigenesis (Wang et al., 1999). Treatment of these cells with PDGF increases the expression of VEGF, and this increase is dependent on PI3K (Wang et al., 1999). PI3K and Akt are also involved in the VEGF-induced up-regulation of intracellular adhesion molecule-1 (ICAM-1; Radisavljevic et al., 2000). Additionally, Akt has been shown to be involved in tumor-induced angiogenesis, an effect mediated through VEGF in conjunction with hypoxia-inducible factor-1α (HIF-1α; Gao et al., 2002). However, the involvement of the PI3K/Akt pathway in the generation of downstream signals for cellular survival induced by VEGF has not been established in vivo. And finally, prevention of radiation induced PI3K/Akt and MAPK signaling impacts upon the cytotoxic effects of this most commonly used form of cancer therapy (Schmidt-Ullrich et al., 2000; Geng et al., 2004; Tan & Hallahan, 2003; Tan et al., 2006; Yacoub et al., 2006).
Another obstacle to designing effective radiotherapy is that there is a poor correlation between cellular responses to ionizing radiation in vitro and in vivo. For example, glioblastoma multiforme (GBM) is insensitive to radiation treatment, and has a universally fatal clinical outcome in both children and adults (Walker et al., 1980; Wallner et al., 1989; Packer, 1999). In vitro studies, however, show that human GBM cell lines exhibit radiosensitivity that is similar to that seen in cell lines derived from more curable human tumors (Allam et al., 1993; Taghian et al., 1993). In accord with the clinical data, the use of in vivo animal models has shown that GBM tumors in vivo are much more radioresistant than the cell lines used to produce them are in vitro (Baumann et al., 1992; Allam et al., 1993; Taghian et al., 1993; Advani et al., 1998; Staba et al., 1998). Thus, the inability to predict the radiosensitivity of a tumor in vivo based upon in vitro experimentation continues to be a significant obstruction to the successful design of radiotherapy treatments of human cancers.
Tumor cells could show enhanced radiosensitivity in vitro compared to in vivo due to the absence of an angiogenic support network in vitro, the presence of which appears to contribute to a tumor's radioresistance in vivo. The response of tumor microvasculature to radiation is dependent upon the dose and time interval after treatment (Kallman et al., 1972; Song et al., 1972; Hilmas & Gillette, 1975; Johnson, 1976; Yamaura et al., 1976; Ting et al., 1991). Tumor blood flow decreases when high doses of radiation in the range of 20 Grays (Gy) to 45 Gy are used (Song et al., 1972). In contrast, blood flow increases when relatively low radiation doses, for example below 500 rads, are administered (Kallman et al., 1972; Hilmas & Gillette, 1975; Johnson, 1976; Yamaura et al., 1976; Gorski et al., 1999). In irradiated mouse sarcomas, for example, blood flow increased during the 3 to 7 days immediately following irradiation (Kallman et al., 1972). Thus, the microvasculature might serve to protect tumor cells from radiation-induced cell death.
Thus, there exists a long-felt need in the art for effective therapies for enhancing the efficacy of radiotherapy, particularly in the context of tumors that are resistant to radiotherapy. To address this need, the presently claimed subject matter provides methods for enhancing the radiosensitivity of cells in a target tissue via administration of an antagonist of a cytosolic phospholipase A2 biological activity.